A Novel Anti-platelet Monoclonal Antibody (3C7) Specific for the Complex of Integrin αIIbβ3 Inhibits Platelet Aggregation and Adhesion*

Activation or ligand binding induces conformational changes in αIIbβ3, resulting in exposure of neoepitopes named ligand-induced binding sites. We reported here a novel monoclonal antibody developed by using Chinese hamster ovary (CHO) cells expressing an activated αIIbβ3 mutant (CHO αIIbβ3Δ717) as the immunogen. This IgG2bκ named 3C7 was specific for the complex of αIIbβ3 as demonstrated by flow cytometry, immunoprecipitation, and EDTA chelating. The binding of 3C7 to platelets increased significantly when platelets were activated by ADP/thrombin or occupied by RGDS peptides, fibrinogen, or PAC-1, suggesting that 3C7 was an anti-ligand-induced binding site antibody. The antibody failed to bind to the CHO cells expressing another αIIbβ3 mutant (β3Y178A) suggesting that the Cys177–Cys184 loop of β3 was likely the epitope for 3C7. 3C7 inhibited platelet aggregation, which was initiated by ADP or thrombin in a dose-dependent manner (IC50s of 5.6 and 0.05 μg/ml, respectively). The antibody also inhibited platelet adhesion to immobilized fibrinogen but not to fibronectin or collagen. These findings suggested that 3C7 was a potent antagonist of integrin αIIbβ3 and a potential anti-thrombotic agent.

Activation or ligand binding induces conformational changes in ␣ IIb ␤ 3 , resulting in exposure of neoepitopes named ligand-induced binding sites. We reported here a novel monoclonal antibody developed by using Chinese hamster ovary (CHO) cells expressing an activated ␣ IIb ␤ 3 mutant (CHO ␣ IIb ␤ 3 ⌬717) as the immunogen. This IgG 2b named 3C7 was specific for the complex of ␣ IIb ␤ 3 as demonstrated by flow cytometry, immunoprecipitation, and EDTA chelating. The binding of 3C7 to platelets increased significantly when platelets were activated by ADP/thrombin or occupied by RGDS peptides, fibrinogen, or PAC-1, suggesting that 3C7 was an antiligand-induced binding site antibody. The antibody failed to bind to the CHO cells expressing another ␣ IIb ␤ 3 mutant (␤ 3 Y178A) suggesting that the Cys 177 -Cys 184 loop of ␤ 3 was likely the epitope for 3C7. 3C7 inhibited platelet aggregation, which was initiated by ADP or thrombin in a dose-dependent manner (IC 50 s of 5.6 and 0.05 g/ml, respectively). The antibody also inhibited platelet adhesion to immobilized fibrinogen but not to fibronectin or collagen. These findings suggested that 3C7 was a potent antagonist of integrin ␣ IIb ␤ 3 and a potential anti-thrombotic agent.
Platelet aggregation and adhesion are the central events in thrombosis and homeostasis (1). Integrin ␣ IIb ␤ 3 (platelet glycoprotein GPIIb/IIIa) is the fibrinogen receptor of platelets. It is in a resting state in the circulation but is activated during platelet aggregation and adhesion. Although the mechanism is not yet fully elucidated, the activation is believed to be induced by conformational changes of ␣ IIb ␤ 3 , resulting in a higher affinity for fibrinogen and other adhesive molecules (2). When stimulated by platelet agonists such as ADP or thrombin, integrin ␣ IIb ␤ 3 undergoes rapid conformational changes, which exposes fibrinogen binding sites and enables the rapid formation of platelet clots (3). Ligand binding to activated integrin ␣ IIb ␤ 3 further induces expression of neoepitopes or ligand-induced binding sites (LIBS) 1 (4,5).
Platelet activation, aggregation, and adhesion are importantly involved in acute coronary syndromes and following certain intravascular therapeutic interventions (6 -9). The central role of integrin ␣ IIb ␤ 3 in thrombosis has led to the development of pharmaceutical agents that block interactions between integrin ␣ IIb ␤ 3 and fibrinogen. The ␣ IIb ␤ 3 antagonists are capable of inhibiting platelet adhesion and aggregation and formation of platelet thrombi at the site of plaque rupture or plaque fissure (10). Because platelet-rich rather than fibrinrich thrombosis was found to be responsible for many acute complications of angioplasty, the blockade of platelet glycoprotein IIb/IIIa receptor was appreciated as valuable in interventional cardiology. Currently there are three integrin ␣ IIb ␤ 3 antagonists used clinically, abciximab (11), tirofiban (12), and eptifibatide (13). Abciximab is a chimeric Fab created based on a murine monoclonal antibody 7E3. Its mechanism of action is thought to be a spatial hindrance of the receptor as opposed to the RGD binding site. Eptifibatide is a synthetic heptapeptide, and tirofiban is a non-peptide antagonist, both mimicking the structure of RGD. All exhibit high affinity binding to integrin ␣ IIb ␤ 3, inhibit ex vivo platelet aggregation, and have proven useful clinically (14).
In the present study, we examined the in vitro anti-platelet effect and binding property of 3C7, a novel monoclonal antibody against the complex of integrin ␣ IIb ␤ 3 raised with CHO cells expressing an activated integrin ␣ IIb ␤ 3 mutant as the immunogen. We found that 3C7 inhibited fibrinogen-mediated platelet aggregation induced by ADP or thrombin. It blocked platelet adhesion to immobilized fibrinogen but not to fibronectin or collagen. The binding of fibrinogen to platelets was partially blocked by 3C7. More interestingly, 3C7 bound to both nonactivated and activated platelets. The binding and affinity increased significantly when platelets were activated by ADP/ thrombin or occupied by ligands for the integrin. To our knowledge, 3C7 is the first anti-LIBS monoclonal antibody specific for the complex of ␣ IIb ␤ 3 .
Production and Characterization of Monoclonal Antibody-The CHO cell line expressing ␣ IIb ␤ 3 ⌬717 was used to immunize BALB/c mice. The splenic lymphocytes from immunized mice were fused with mouse myeloma FO cells as described previously (15). Hybridoma culture supernatants were screened for antibodies reactive with integrin ␣ IIb ␤ 3 using cellular enzyme-linked immunosorbent assay. One positive hybridoma, named 3C7, was subcloned twice by limiting dilution. The monoclonal antibody was then produced in mouse ascetic fluid and purified with immobilized protein G. The 3C7 subtyping was performed using a mouse immunoglobulin subtype identification kit (Hycult Biotech, Uden, Netherlands). The affinity of 3C7 for ␣ IIb ␤ 3 of intact platelets was measured essentially as described previously (5) with an exception that 3C7 was labeled with sulfo-NHS-LC-biotin instead of 125 I. Briefly, platelets were isolated and washed as described previously (16). Then washed platelets (3 ϫ 10 8 /ml) were incubated with increasing amounts of labeled 3C7 at room temperature for 1 h. In some cases, platelets were exposed to 1 mM RGDS or 0.1 mM ADP before addition of 3C7. To determine the amount of total 3C7 binding, the free 3C7 in the supernatant was measured with a quantitative enzyme-linked immunosorbent assay using avidin coated on microtiter wells and subtracted from the total amount added. Nonspecific binding was determined in the presence of a 50-fold excess of unlabeled antibody. The K d value was obtained using the Scatchard Plot.
Immunoprecipitation and Western Blot-For immunoprecipitation, cell lysates were first incubated overnight at 4°C with 3C7 or SZ21 and then with protein A-Sepharose beads for another 2 h. The beads were washed with the lysis buffer six times and then boiled in the SDS sample buffer (2% SDS, 10% glycerol, 25 g/ml bromphenol blue in 15.63 mM Tris-Cl, pH 6.8). For Western blot analysis, platelet or cell lysates were first centrifuged at 10,000 rpm for 10 min at 4°C before their protein concentrations were determined. Lysates or immunoprecipitates were applied for a 7.5% SDS-PAGE under both non-reducing and reducing conditions and then transferred to a Hybond C nitrocellulose membrane. The membrane was blocked and probed for 2 h at room temperature with SZ21 and SZ22. After several washes, the membrane was incubated with the secondary goat anti-mouse IgG conjugated to horseradish peroxidase and finally developed using the chemiluminescence ECL kit.
Flow Cytometry Analysis-Binding of antibodies to platelets and CHO cells expressing integrins were analyzed by flow cytometry as previous described (16). In brief, washed platelets (1ϫ10 6 /ml) or cells (5ϫ10 5 /ml) were incubated with the primary antibody for 30 min at 4°C followed by incubation with FITC-conjugated donkey anti-mouse IgG and analyzed on a FACScan (Becton Dickinson). For some experiments, washed platelets were exposed to different treatments prior to incubation with 3C7. To assess the effect of the separate subunit (␣ IIb or ␤ 3 ), CHO cells expressing ␣ IIb ␤ 3 were firstly treated by incubation with 5 mM EDTA at 37°C or room temperature for 30 min. To study the effect of the activation state of ␣ IIb ␤ 3 , washed platelets were pretreated with 0.1 mM ADP or 0.5 units/ml thrombin at room temperature for 20 min.
To study the effect of ligand occupation, washed platelets were incubated directly with 1 mM RGDS at room temperature for 20 min or with fibrinogen or PAC-1 for 45 min after platelets were activated with 0.1 mM ADP or 0.5 units/ml thrombin before addition of 3C7. Finally, to investigate the effect of 3C7 on fibrinogen binding, platelets were activated with 0.1 mM ADP, then incubated with 300 g/ml FITC-fibrinogen in the presence of various concentrations of 3C7 for 15 min at 37°C and analyzed by flow cytometry (17).
Platelet Aggregation and Adhesion-Platelet aggregation was performed using citrated platelet-rich plasma. After incubation at 37°C for 5 min with various concentrations of 3C7, aggregation was initiated by adding 10 M ADP or 0.35 units/ml thrombin. The maximal platelet aggregation within 5 min was recorded using an aggregometer. Platelet adhesion to immobilized ligand was done as described previously (18) with one modification; platelets were labeled with sulfo-NHS-LC-Biotin instead of 51 Cr. Briefly, 2 ϫ 10 7 labeled platelets were incubated with various concentrations of 3C7 and then added to each well coated with fibrinogen, fibronectin, or collagen and allowed to adhere at 37°C for 1 h. The non-adherent platelets were aspirated off before the addition of horseradish peroxidase-conjugated streptavidin to be developed with the TMB substrate. The extent of adhesion at each concentration of 3C7 was detected by comparison with the standard curve and expressed as the percentage of the control in which platelets were not preincubated with 3C7.

Generation and Characterization of Monoclonal Antibody-
Monoclonal antibodies were produced by fusing FO cells with splenocytes from mice immunized with CHO cells expressing ␣ IIb ␤ 3 ⌬717, an ␣ IIb ␤ 3 mutant with an active conformation (19). Ten days after fusion, 80% of hybridoma supernatants were positive in enzyme-linked immunosorbent assay against CHO/ ␣ IIb ␤ 3 ⌬717 cells. Specific clones were further screened for their reactivity with ␣ IIb ␤ 3 . The antibody produced by a positive clone (named 3C7) was identified as IgG 2b -immunoglobulin and purified by an affinity column of protein G. Flow cytometry analysis revealed that 3C7 had no binding to CHO cells expressing either ␤ 3 or ␣ IIb alone, or ␣ V ␤ 3 . In contrast, 3C7 recognized ␣ IIb ␤ 3 expressed on CHO cells or platelets (Fig. 1). 3C7 was unable to probe ␣ IIb ␤ 3 reduced or non-reduced in Western blot (data dot shown).
3C7 Is Specific for ␣ IIb ␤ 3 Complex-Because ␤ 3 is required for the post-translation process of ␣ IIb (20), the lack of 3C7 binding to ␣ IIb transfected CHO cells (Fig. 1B) is insufficient to conclude that 3C7 does not recognize ␣ IIb . Therefore, we used immunoprecipitation to confirm this property of 3C7. As shown in Fig. 2, although anti-␤ 3 mAb SZ21 precipitated ␤ 3 from the lysate of CHO/␣ V ␤ 3 , 3C7 failed to precipitate ␣ IIb or ␤ 3 using the lysate of CHO/␣ IIb or CHO/␣ V ␤ 3, suggesting that it was not against ␣ IIb or ␣ V . By contrast, two bands corresponding to ␣ IIb and ␤ 3 were found in the 3C7 immunoprecipitation against the lysate of platelets or CHO/␣ IIb ␤ 3 (Fig. 2). Additionally, 3C7 was confirmed to be specific for intact ␣ IIb ␤ 3 using EDTA chelating. The CHO cells expressing the complex of ␣ IIb ␤ 3 were firstly treated with 5 mM EDTA at 37°C for 30 min, which was proved to dissociate the complex of ␣ IIb ␤ 3 (21), and then binding of 3C7 was assayed by flow cytometry. As a control, the cells were preincubated with 5 mM EDTA at room temperature for 30 min, which is known not to affect the structure of the complex. As shown in Fig. 3, the binding of 3C7 to the cells pretreated with 5 mM EDTA at 37°C was diminished to the level of background. In contrast, binding of 3C7 to the control cells was not affected as compared with the cells without any treatment.
3C7 Recognizes a LIBS Epitope on Integrin ␣ IIb ␤ 3 in Platelets-Because 3C7 was raised by immunizing mice with CHO cells expressing the activated state of ␣ IIb ␤ 3 (␣ IIb ␤ 3 ⌬717), its binding properties to activated platelets were therefore consequently investigated. First, using platelets activated by ADP or thrombin, 3C7 binding was increased by more than 5-fold (Fig.  4A). Second, 3C7 binding was also increased after the addition of RGDS, fibrinogen or PAC-1 (Fig. 4B). The binding affinity of 3C7 to platelets was also measured in the presence of different platelet ligands. The K d for resting platelets and for platelets activated by ADP or occupied with RGDS were 4.60 Ϯ 2.13, 1.11 Ϯ 0.99, and 1.30 Ϯ 1.22 nM (n ϭ 3), respectively. These data indicated that 3C7 recognized a LIBS epitope. It was reported that certain anti-LIBS antibodies activated ␣ IIb ␤ 3 and promoted fibrinogen binding to platelets (22,23). However, 3C7 was not an activating antibody (Fig. 4, C and D).
Platelet Aggregation Initiated by ADP or Thrombin Was Blocked by 3C7-3C7 inhibited platelet aggregation induced by ADP or thrombin in a dose-dependent manner with an IC 50 value of 5.6 g/ml for ADP and 0.05 g/ml for thrombin, respectively (Fig. 5).
Platelet Adhesion to Immobilized Fibrinogen Was Inhibited by 3C7-␣ IIb ␤ 3 is critical for platelet adhesion to immobilized ligands. 3C7 inhibited platelet adhesion to immobilized fibrinogen in a dose-dependent manner with an IC 50 of 0.9 g/ml (Fig. 6). In contrast, 3C7 had no effect on platelet adhesion to either immobilized fibronectin (Fig. 6) or collagen (data not shown).
3C7 Inhibited Fibrinogen Binding to Platelets-Using FITClabeled fibrinogen, binding of fibrinogen to ADP-stimulated platelets was measured with FACS in the presence of 3C7. The IC 50 was measured as 0.3 g/ml for ADP, and the maximum inhibition (65%) was obtained at 1.0 g/ml that was a considerably low concentration for an antibody antagonist (Fig. 7).
␤ 3 Y178 Was Involved in the Antigen Epitope for 3C7-It is known that the Cys 177 -Cys 184 loop of ␤ 3 is important for c7E3 (abciximab) binding as well as ligand binding (24). After carefully examining sequences of the Cys 177 -Cys 184 loop from different species, we found that Tyr 178 was absolutely conservative. Therefore, Tyr 178 was mutated (␤ 3 Y178A and ␤ 3 Y178I) and co-expressed with ␣ IIb in CHO to investigate the possibility of the loop also being the epitope for 3C7. As shown in Fig. 8, the expression of ␤ 3 was monitored using SZ21 (specific for ␤ 3 ) and was unaffected by the mutation of ␤ 3 Y178A or ␤ 3 Y178I. However, 3C7 had a full binding to ␣ IIb ␤ 3 Y178I and nearly no binding to ␣ IIb ␤ 3 Y178A, indicating that the Cys 177 -Cys 184 loop of ␤ 3 was likely to be the epitope for 3C7, and the hydrophobicity of Y178 was crucial for its function. DISCUSSION Three types of anti-LIBS antibodies against ␣ IIb or ␤ 3 are described in the literature (a) with no effect on platelet aggregation (PMI-1, ␣ IIb -specific) (25,26), (b) with inhibition of platelet aggregation (anti-LIBS-1, ␤ 3 -specific) (4), and (c) with activation of ␣ IIb ␤ 3 (D3GP3, anti-LIBS-2, -3, -6, ␤ 3specific) (4,5,22). To our knowledge, 3C7 is the first anti-LIBS mAb against the complex of ␣ IIb ␤ 3 . It has a higher affinity for active platelets and inhibits both platelet aggregation and adhesion. Moreover, its binding to platelets is enhanced by additional ligand binding.
These differences may be attributed to our new approach, which used CHO cells expressing activated ␣ IIb ␤ 3 as the immunogen. This was based on the assumption that ␣ IIb ␤ 3 with different conformations could be expressed on CHO cells where it functioned similar to its counterparts in platelets. Our data confirmed that deletion of the ␤ 3 cytoplasmic domain induced conformational changes in the extracellular part of ␣ IIb ␤ 3 and an exposure of neoepitope LIBS (19). Interestingly, 3C7 recognized the complex of ␣ IIb ␤ 3 exclusively and did not react with ␣ IIb or ␤ 3 . This was demonstrated by EDTA chelating, which caused dissociation of the complex. Our strategy allowed recognition of epitopes not exposed or inaccessible on the resting integrin. In contrast, all other anti-LIBS or activation-dependent mAbs were produced using either whole platelets (27),  6. Effect of 3C7 on human platelet adhesion to immobilized ligands. Biotinylated platelets were incubated with various concentrations of 3C7 and then allowed to adhere to wells coated with indicated concentrations of fibrinogen (diamonds) and fibronectin (squares). After non-adherent platelets were removed by washing, adhered platelets were quantitated using horseradish peroxidase-conjugated streptavidin. The extent of adhesion was expressed as the percentage of control platelets adhered without preincubation with 3C7. Data were mean Ϯ S.D. of at least three different determinations. platelet membranes (26,28), purified ␣ IIb ␤ 3 (5), purified ␣ IIb ␤ 3 mixed with RGDS (4), or synthetic ␣ IIb ␤ 3 peptides (29) as the immunogen. In addition, a single-chain variable fragment specific for activated ␣ IIb ␤ 3 was obtained using phage display against activated platelets (30).
Efforts to raise activation-dependent antibodies using whole platelets activated prior to immunization encountered a number of technique difficulties. Our experiments demonstrated an effective approach to solve the problem. First, transfected cells preserve native conformations of the integrin. Second, it allows production of antibodies against different conformations of the integrin specifically made with site-directed mutations.
The disulfide-bonded loop (Cys 187 -Cys 193 of ␤ 1 , Cys 169 -Cys 176 of ␤ 2 , and Cys 177 -Cys 184 of ␤ 3 ) laying on the upper surface of the ␤ I domain and projecting from the surface containing sites adjacent to a metal ion-dependent adhesion site (31) is known to be critical for affinity and specificity of receptor-ligand binding. Replacement of the ␤ 2 loop with the ␤ 3 loop activates binding of ␣ L ␤ 2 to ICAM-1 (32). The mutation of ␤ 1 integrin (T188I) at this loop promotes cell spreading in human SCC4 keratinocyte (33). This loop also determines the differential regulation of Rho GTPases by ␤ 1 and ␤ 3 integrins in intracellular signaling events (34). Moreover, ligand binding sites are found close to or overlapping with residues at this loop by epitope mapping with ligand mimetic mAbs (24). The disruption of the Cys 177 -Cys 184 disulfide bond of ␤ 3 affects binding of mAb (AP2, LM609, or c7E3) (35) as well as exposure of the LIBS epitope (35). In this report, we have demonstrated that the Cys 177 -Cys 184 loop of ␤ 3 is also important for 3C7 recognizing a unique epitope for the complex of ␣ IIb ␤ 3 . It further confirms the structural importance of this disulfidebonded loop for its role in antibody and ligand binding.
3C7 preferentially binds to platelets stimulated by ADP or thrombin in vitro. Under the condition of our assay, platelet aggregation stimulated by ADP or thrombin was inhibited with a very different IC 50 . Both ADP (36) and thrombin (37) activate platelets via different G-protein-coupled receptors and lead to distinct structural rearrangements of ␣ IIb ␤ 3 (38) . This may explain the discrepant IC 50 of 3C7 for its inhibitory effect on ADP or thrombin-induced platelet aggregation. 3C7 inhibits platelet adhesion to immobilized fibrinogen but not to fibronectin or collagen. This may be because of the fact that platelet adhesion to fibronectin and collagen is also mediated by other integrins such as ␣ V ␤ 3 , ␣ 3 ␤ 1 , ␣ 5 ␤ 1 , and ␣ 2 ␤ 1 (39).
Although it is unclear how ligands interact with ␣ IIb ␤ 3 , it is suggested that native ligands (e.g. fibrinogen) or ligand-mimetic antibodies may directly contact discontinuous binding sites at both subunits, which may constitute a ligand binding pocket (24). Three human ␣ IIb ␤ 3 -specific mAbs (PAC-1 (27), OP-G2 (40), and LJ-CP3 (41)) have the RYD sequence that mimics RGD in their CDR3 regions, indicating that the RYD sequence may occupy the same space as RGD does (42). Although 3C7 inhibits fibrinogen binding to platelets, its binding to platelets is promoted by pretreatment of fibrinogen or RGDS. Therefore, the inhibitory effect of 3C7 seems not because of a direct occupancy of the RGDS binding site. However, when 3C7 binds, it blocks fibrinogen binding to platelets probably because of its spatial effect. ␣ IIb ␤ 3 antagonists currently available either bind to the resting integrin or induce a transition of ␣ IIb ␤ 3 from a resting to ligand competent state (43). This has been implicated in thrombocytopenia occasionally reported after administration of such antagonistsincludingabciximabinman (44,45).Thus,activationdependent mAbs would be of great interest to develop for therapeutic use. Previous studies suggested that agents recognizing ␣ IIb ␤ 3 with high affinity and selectivity for the activated rather than resting integrin possess a high therapeutic potential for thromboembolic events (46). Activation-dependent antibodies mimicking fibrinogen may also alleviate certain adverse effects such as increased risk of bleeding encountered with abciximab in a clinic (44). Additionally, antibodies preferentially recognizing ligand-occupied conformations may be of value in diagnosis. In comparison with abciximab, 3C7 is comparable for its binding affinity and inhibitory activity to platelets but is highly specific for the complex of ␣ IIb ␤ 3 . The clinical potential of the unique specificity of 3C7 needs to be evaluated in vivo with animal studies, because ␣ V ␤ 3 , the spare target of abciximab, is widely distributed in diverse tissues and involved in many physiological and pathological events.